Combinations and uses thereof

ABSTRACT

The present disclosure describes a pharmaceutical combination of an anti-CD38 antibody and lenalidomide and a pharmaceutical combination of an anti-CD38 antibody and bortezomib.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/825,325, filed Mar. 21, 2013, which is a national stage entry ofInternational Patent Application No. PCT/EP2011/066648, filed Sep. 26,2011, which claims the benefit of U.S. Provisional Patent ApplicationNos. 61/486,814, filed May 17, 2011; 61/468,607, filed Mar. 29, 2011;61/437,696, filed Jan. 31, 2011; and 61/386,619, filed Sep. 27, 2010,and claims foreign priority benefit of European Patent Application No.10180485.4, filed Sep. 27, 2010, which are all incorporated herein byreference in their entireties.

BACKGROUND

Multiple myeloma is a B cell malignancy characterized by the latentaccumulation in bone marrow of secretory plasma cells with a lowproliferative index and an extended life span. The disease ultimatelyattacks bones and bone marrow, resulting in multiple tumors and lesionsthroughout the skeletal system.

Approximately 1% of all cancers, and slightly more than 10% of allhematologic malignancies, can be attributed to multiple myeloma (MM).The incidence of MM increases in the aging population, with the medianage at time of diagnosis being about 61 years. The currently availabletherapies for multiple myeloma include chemotherapy, stem celltransplantation, Thalomid® (thalidomide), Velcade® (bortezomib), Aredia®(pamidronate), and Zometa® (zoledronic acid). The current treatmentprotocols, which include a combination of chemotherapeutic agents suchas vincristine. BCNU, melphalan, cyclophosphamide, adriamycin, andprednisone or dexamethasone, yield a complete remission rate of onlyabout 5%, and median survival is approximately 36-48 months from thetime of diagnosis. Recent advances using high dose chemotherapy followedby autologous bone marrow or peripheral blood mononuclear celltransplantation have increased the complete remission rate and remissionduration. Yet overall survival has only been slightly prolonged, and noevidence for a cure has been obtained. Ultimately. MM patients oftenrelapse, even under maintenance therapy with interferon-alpha (IFN-α)alone or in combination with steroids.

Non-Hodgkin's lymphoma is a broad classification of lymphomas, which arecancers originating from the lymphatic system when lymphocytes (B-cellsor T-cells) become malignant and proliferate uncontrollably to form atumor mass. In total NHL encompasses around 30 different subtypes oflymphoma, including Diffuse large B-cell lymphoma (DLBCL) and follicularlymphoma (FL). The incidence of NHL will reach over 140,000 in the majormarkets by 2019. The available treatment options includeRituxan/MabThera, combinations thereof, such as, R-CHOP (rituximab,cyclophosphamide, doxorubicin, vincristine and prednisone), R-CVP(Rituxan, cyclophosphamide, vincristine and prednisone), andchemotherapy. In addition, following remission or after relapse,hematopoietic stem cell transplantation may be considered. Despite thecurrent treatment options, however, the survival rates within high riskgroups of aggressive NHL can be as low as 30% over 5 years. Therefore,there remains a high unmet need for effective treatments and combinationtreatments.

CD38 is an example of an antigen expressed on such malignant plasmacells, and other lymphocytes. Functions ascribed to CD38 include bothreceptor mediation in adhesion and signaling events and (ecto-)enzymatic activity. As an ectoenzyme, CD38 uses NAD+ as substrate forthe formation of cyclic ADP-ribose (cADPR) and ADPR, but also ofnicotinamide and nicotinic acid-adenine dinucleotide phosphate (NAADP).cADPR and NAADP have been shown to act as second messengers for Ca2+mobilization. By converting NAD+ to cADPR, CD38 regulates theextracellular NAD+ concentration and hence cell survival by modulationof NAD-induced cell death (NCID). In addition to signaling via Ca2+,CD38 signaling occurs via cross-talk with antigen-receptor complexes onT and B cells or other types of receptor complexes, e.g. MHC molecules,and is in this way involved in several cellular responses, but also inswitching and secretion of IgG.

Antibodies specific for CD38 are described in WO1999/62526 (MayoFoundation); WO200206347 (Crucell Holland); US2002164788 (JonathanEllis) which is incorporated by reference in its entirety; WO2005/103083(MorphoSys AG), U.S. Ser. No. 10/588,568, which is incorporated byreference in its entirety; WO2006/125640 (MorphoSys AG), U.S. Ser. No.11/920,830, which is incorporated by reference in its entirety, andWO2007/042309 (MorphoSys AG), U.S. Ser. No. 12/089,806, which isincorporated by reference in its entirety; WO2006099875 (Genmab), U.S.Ser. No. 11/886,932, which is incorporated by reference in its entirety;and WO08/047,242 (Sanofi-Aventis), U.S. Ser. No. 12/441,466, which isincorporated by reference in its entirety.

Combinations of antibodies specific for CD38 and other agents aredescribed in WO200040265 (Research Development Foundation); WO2006099875and WO2008037257 (Genmab); and WO2010061360, WO2010061359, WO2010061358and WO2010061357 (Sanofi Aventis), which are all incorporated byreference in their entireties.

It is clear that in spite of the recent progress in the discovery anddevelopment of anti-cancer agents, many forms of cancer involvingCD38-expressing tumors still have a poor prognosis. Thus, there is aneed for improved methods for treating such forms of cancer.

SUMMARY

In one aspect, the present disclosure relates to a synergisticcombination of an antibody specific for CD38 and thalidomide or ananalog thereof, e.g. lenalidomide. In another aspect the presentdisclosure relates to a synergistic combination comprising an antibodyspecific for CD38 and bortezomib or other proteasome inhibitor. Suchcombinations are useful in the treatment of cancers, such as, multiplemyeloma and/or non-Hodgkin's lymphoma.

In vitro and in vivo models are considered predictive of how a certaincompound or combination of compounds would behave in humans. Here, thecombinations of an antibody specific for CD38 and lenalidomide wastested in human multiple myeloma cell lines and synergy was identified.In addition the combination of an antibody specific for CD38 andlenalidomide, and a combination of an antibody specific for CD38 andbortezomib were tested in mouse models against both multiple myelomacells and Burkitt's lymphoma (a form of NHL) cells and synergy wasidentified. Therefore, the combinations will be effective in thetreatment of humans in multiple myeloma and/or non-Hodgkin's lymphoma.In addition, the antibody specific to CD38 exemplified in the presentspecification is entering into clinical trials, where such combinationscan be confirmed in humans.

When compounds are combined either in vitro or in vivo, one expects thatthe combination has only additive effects. Quite unexpectedly, theinventors found that the combination of a particular anti-CD38 antibodyand lenalidomide mediated a synergistic level of Antibody-DependentCell-Mediated Cytotoxicity (ADCC) in both the AMO-1 and NCI-H929multiple myeloma cell lines. In addition, and also unexpectedly, aparticular anti-CD38 antibody when combined with lenalidomide or whencombined with bortezomib mediated a synergistic level of reduction inbone lysis in the NCI-H929 SCID mouse model and synergisticallyincreased the median survival days in the RAMOS SCID mouse model.Therefore, both the combination of the exemplified antibody specific forCD38 and lenalidomide and the exemplified antibody specific for CD38 andbortezomib behaved synergistically in the in vitro and/or in vivo modelsrelevant to multiple myeloma and/or non-Hodgkin's lymphoma.

An aspect of the present disclosure comprises a combination wherein theantibody specific for CD38 comprises an HCDR1 region of sequenceGFTFSSYYMN (SEQ ID NO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2region of sequence GISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region ofsequence DLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequenceSGDNLRHYYVY (SEQ ID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ IDNO: 5), and an LCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) and thethalidomide or an analog thereof is lenalidomide. In preferred aspects,the combination is used for the treatment of multiple myeloma and/ornon-Hodgkin's lymphoma.

An aspect of the present disclosure comprises a combination wherein theantibody specific for CD38 comprises an HCDR1 region of sequenceGFTFSSYYMN (SEQ ID NO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2region of sequence GISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region ofsequence DLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequenceSGDNLRHYYVY (SEQ ID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ IDNO: 5), and an LCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) and theproteasome inhibitor is bortezomib. In preferred aspects, thecombination is used for the treatment of multiple myeloma and/ornon-Hodgkin's lymphoma.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the effects of lenalidomide alone on the expression of CD38in AMO-1 cells.

FIG. 2 shows the effects of lenalidomide alone on cell proliferation invarious multiple myeloma cell lines. This measure represents therelative cytoxicity of lenalidomide on each cell line.

FIG. 3 shows the mediation of ADCC on AMO-1 cells by the combination ofMOR03087 and lenalidomide. The PBMCs and AMO-1 cells were treated withlenalidomide prior to treatment with MOR03087. MOR03207 binds lysosyme,and is used as isotype control, as it is IgG1. LEN representslenalidomide. “Theoretical” represents the addition of the value ofMOR03087 alone and the value of LEN alone. The data shown are theaverages from Table 3b.

FIG. 4 shows the mediation of ADCC on AMO-1 cells by the combination ofMOR03087 and lenalidomide. Only the PBMCs were treated with lenalidomideprior to treatment with MOR03087. MOR03207 binds lysosyme, and is usedas isotype control, as it is IgG1. LEN represents lenalidomide.“Theoretical” represents the addition of the value of MOR03087 alone andthe value of LEN alone. The data shown are the averages from Table 4b.

FIG. 5 shows the effects of lenalidomide alone on the expression of CD38in NCI-H929 cells.

FIG. 6 shows the mediation of ADCC on NCI-H929 cells by the combinationof MOR03087 and lenalidomide. The PBMCs and NCI-H929 cells were treatedwith lenalidomide prior to treatment with MOR03087. Theoreticalrepresent the combination calculated using the fractional productconcept of Chou et al. The data shown are the averages from Table 7b.

FIG. 7 shows the mediation of ADCC on NCI-H929 cells by the combinationof MOR03087 and lenalidomide. Only the PBMCs were treated withlenalidomide prior to treatment with MOR03087. Theoretical represent thecombination calculated using the fractional product concept of Chou etal. The data shown are the averages from Table 8b.

FIG. 8 shows the growth inhibition of various multiple myeloma celllines caused by bortezomib alone. The IC50 on AMO-1 cells was 3.9 nM.The IC50 on LP-1 cells was 6.1 nM. The IC50 on NCI-H929 cells was 3.3nM. The IC50 on RPMI-8226 cells was 9.0 nM.

FIG. 9 shows the mediation of ADCC on NCI-H929 cells by the combinationof MOR03087 at 15 μg/ml and Velcade® (bortezomib). The two chartsrepresent two different donors.

FIG. 10 shows the mediation of ADCC on LP-1 cells by the combination ofMOR202 at 15 μg/ml and Velcade® (bortezomib). The two charts representtwo different donors.

FIG. 11 shows the amino acid sequence of MOR202.

FIG. 12 shows the Best Fit curve, as described in Chou et al., of theMOR202 and lenalidomide combination in the mediation of ADCC on AMO-1cells and it is also representative for the Best Fit curve generated foranalysis of the mediation of ADCC on NCI-H929 cells.

FIGS. 13-18 show the Chou factor synergy analysis for six separateexperiments using the combination of MOR202 and lenalidomide in themediation of ADCC on AMO-1 cells. FIG. 13 shows experiment 1. FIG. 14shows experiment 2. FIG. 15 shows experiment 3. FIG. 16 shows experiment4. FIG. 17 shows experiment 5. FIG. 18 shows experiment 6. FIGS. 13-15were derived from the three experiments shown in Tables 3a-c, and FIG.3. FIGS. 16-18 were derived from the three experiments shown in Tables4a-c, and FIG. 4.

FIG. 19 shows the MicroCT Scan mean total bone volume of each of thestudy groups described in Example 7, where the results are shown inTable 11.

FIG. 20 shows the MicroCT Scan mean total bone volume of each of thestudy groups described in Example 11, where the results are shown inTable 17.

DETAILED DESCRIPTION OF THE INVENTION

“Synergy”, “synergism” or “synergistic” mean more than the expectedadditive effect of a combination. The “synergy”, “synergism” or“synergistic” effect of a combination is determined herein by themethods of Chou et al., and/or Clarke et al. See Ting-Chao Chou,Theoretical Basis, Experimental Design, and Computerized Simulation ofSynergism and Antagonism in Drug Combination Studies, Pharmacol Rev58:621-681 (2006), which is incorporated by reference in its entirety.In Chou et al., multiple methods of determining synergism are disclosedand at least one of these methods is used herein. See also Clarke etal., Issues in experimental design and endpoint analysis in the study ofexperimental cytotoxic agents in vivo in breast cancer and other models,Breast Cancer Research and Treatment 46:255-278 (1997), which isincorporated by reference in its entirety.

The term “antibody” means monoclonal antibodies, including any isotype,such as, IgG, IgM, IgA, IgD and IgE. An IgG antibody is comprised of twoidentical heavy chains and two identical light chains that are joined bydisulfide bonds. Each heavy and light chain contains a constant regionand a variable region. Each variable region contains three segmentscalled “complementarity-determining regions” (“CDRs”) or “hypervariableregions”, which are primarily responsible for binding an epitope of anantigen. They are referred to as CDR1, CDR2, and CDR3, numberedsequentially from the N-terminus. The more highly conserved portions ofthe variable regions outside of the CDRs are called the “frameworkregions”. An “antibody fragment” means an Fv, scFv, dsFv, Fab, Fab′F(ab′)2 fragment, or other fragment, which contains at least onevariable heavy or variable light chain, each containing CDRs andframework regions.

THALOMID® (thalidomide) in combination with dexamethasone is indicatedfor the treatment of patients with newly diagnosed multiple myeloma, andis marketed by Celgene.

A “thalidomide analog” includes, but is not limited to, thalidomideitself, lenalidomide (CC-5013, Revlimid™), Pomalidomide (CC4047,Actimid™) and the compounds disclosed in WO2002068414 and WO2005016326,which are incorporated by reference in their entireties. The term refersto a synthetic chemical compound using the thalidomide structure as abackbone (e.g., side groups have been added or such groups have beendeleted from the parent structure). The analog differs in structure fromthalidomide and its metabolite compounds such as by a difference in thelength of an alkyl chain, a molecular fragment, by one or morefunctional groups, or a change in ionization. The term “thalidomideanalog” also includes the metabolites of thalidomide. Thalidomideanalogs include the racemic mixture of the S- and the R-enantiomer of arespective compound and the S-enantiomer or to the R-enantiomerindividually. The racemic mixture is preferred. Thalidomide analogsinclude the compounds of the following structures:

wherein R21, R22, R23, and R24 are each independently H, alkoxy, amino,or alkylamine, and

wherein R21, R22, R23, and R24 are each independently H, alkoxy, amino,or alkylamine. Lenalidomide is currently marketed as Revlimid® byCelgene for the treatment of multiple myeloma. Lenalidomide is describedas having at least the following properties in relation to the treatmentof tumors, a) cytotoxic to tumor cells, Gandhi et al., Lenalidomideinhibits proliferation of Namalwa CSN.70 cells and interferes with Gab1phosphorylation and adaptor protein complex assembly, Leuk Res.,30(7):849-58 (2006), which is incorporated by reference in its entirety;b) activates natural killer (Nk) cells, Gandhi et al., Dexamethasonesynergizes with lenalidomide to inhibit multiple myeloma tumor growth,but reduces lenalidomide-induced immunomodulation of T and NK cellfunction, Curr Cancer Drug Targets, 1; 10(2):155-67 (March 2010), whichis incorporated by reference in its entirety; and c) upregulates CD38expression on tumor cells, See Lapalombella et al., Lenalidomidedown-regulates the CD20 antigen and antagonizes direct andantibody-dependent cellular cytotoxicity of rituximab on primary chroniclymphocytic leukemia cells, Blood, 112:13, 5180-5189 (15 Dec. 2008),which is incorporated by reference in its entirety. “LEN” is used todescribe lenalidomide.

As described, thalidomide analogs upregulate the expression of CD38 ontumor cells. Other agents that upregulate the expression of CD38 on thesurface of tumor cells are described in WO00/40265, U.S. Ser. No.09/226,895, which is incorporated by reference in its entirety (ResearchDevelopment Foundation).

A “proteasome inhibitor” refers to a compound that blocks the action ofproteasomes, i.e. cellular complexes that break down proteins, such asfor example the p53 protein. Several classes of proteasome inhibitorsare known. The class of the peptide boronates includes bortezomib (INN,PS-341; Velcade®), a compounds which is approved in the U.S. for thetreatment of relapsed multiple myeloma. Another peptide boronate isCEP-18770. Other classes of proteasome inhibitors include peptidealdehydes (e.g. MG132), peptide vinyl sulfones, peptide epoxyketones(e.g. epoxomicin, carfilzomib), β lactone inhibitors (e.g. lactacystin,MLN 519, NPI-0052, Salinosporamide A), compounds which createdithiocarbamate complexes with metals (e.g. Disulfuram, a drug which isalso used for the treatment of chronic alcoholism), and certainantioxidants (e.g. Epigallocatechin-3-gallate) catechin-3-gallate, andSalinosporamide A.

“VH” refers to the variable region of an immunoglobulin heavy chain ofan antibody, or antibody fragment. “VL” refers to the variable region ofthe immunoglobulin light chain of an antibody, or antibody fragment.

The term “CD38” refers to the protein known as CD38, having thefollowing synonyms: ADP-ribosyl cyclase 1, cADPr hydrolase 1, CyclicADP-ribose hydrolase 1, T10.

Human CD38 has the amino acid sequence of:

(SEQ ID NO: 7) MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDS SCTSEI.

“MOR202” an anti-CD38 antibody whose amino acid sequence is provided inFIG. 11. “MOR202” and “MOR03087” are used as synonyms to describe theantibody shown in FIG. 11.

The DNA sequence encoding the MOR202 Variable Heavy Domain is:

(SEQ ID NO: 12) CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTTCTTCTTATTATATGAATTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGGTATCTCTGGTGATCCTAGCAATACCTATTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGATCTTCCTCTTGTTTATACTGGTTTTGCTTATTGGGGCCAAGGCACCC TGGTGACGGTTAGCTCA

The DNA sequence encoding the MOR202 Variable Light Domain is:

(SEQ ID NO: 13) GATATCGAACTGACCCAGCCGCCTTCAGTGAGCGTTGCACCAGGTCAGACCGCGCGTATCTCGTGTAGCGGCGATAATCTTCGTCATTATTATGTTTATTGGTACCAGCAGAAACCCGGGCAGGCGCCAGTTCTTGTGATTTATGGTGATTCTAAGCGTCCCTCAGGCATCCCGGAACGCTTTAGCGGATCCAACAGCGGCAACACCGCGACCCTGACCATTAGCGGCACTCAGGCGGAAGACGAAGCGGATTATTATTGCCAGACTTATACTGGTGGTGCTTCTCTTGTGTTTGGCGGCGGCACGAAGTTAACCGTTCTTGGCCAG

Antibody “Ref mAB5” is an anti-CD38 antibody whose amino acid sequenceis provided below (the CDRs are bolded and underlined):

VH: (SEQ ID NO: 21) QVQLVQSGAEVAKPGTSVKLSCKASGYTFT DYWMQ WVKQRPGQGLEWIGTIYPGDGDTGYAQKFQG KATLTADKSSKTVYMHLSSLASEDSAVYYCAR GDYYGSNSLDYWGQGTSVTVSS VL: (SEQ ID NO: 22) DIVMTQSHLSMSTSLGDPVSITC KASQDVSTVVAWYQQKPGQSPRRLIY SASYRYI GVPDRFTGSGAGTDFTFTISSVQAEDLAVYYC QQHYSPPYT FGGGTKLEIKRT

The CDRs of Ref mAB5 are defined by Kabat et al. and an antibody havingthe same CDRs as Ref mAB5 is described in WO2008/047242, U.S. Ser. No.12/441,466, which is incorporated by reference in its entirety.

“Fc region” means the constant region of an antibody, which in humansmay be of the IgG1, 2, 3, 4 subclass or others. The sequences of humanFc regions are available at IMGT, Human IGH C-REGIONs,www.imgt.org/IMGTrepertoire/Proteins/protein/human/IGH/IGHC/Hu_IGHCallgenes.html(retrieved on 16 May 2011).

“Enhances ADCC activity” means an increase in the mediation of antibodydependent cell-mediated cytotoxicity. Amino acid modifications withinthe Fc region that result in an enhacement of ADCC activity aredisclosed in WO200042072 Genentech, WO2004029207A2 Xencor, andWO2004063351A2 Macrogenics, which are all incorporated by reference intheir entireties.

“MOR03207” is an antibody whose amino acid sequence is:

VH: (SEQ ID NO: 8) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSS VL: (SEQ ID NO: 9)DIELTQPPSVSVAPGQTARISCSGDNLPAYTVTWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCASWDPSSGVVF GGGTKLTVLGQ.

MOR03207 binds lysosyme, and is used as isotype control, as it is IgG1.

A “combination” means more than one item, e.g. a compound such as anantibody and lenalidomide.

The present disclosure also relates to combinations, pharmaceuticals,and pharmaceutical compositions containing the described combinations.The two components of the synergistic combination of the presentinvention, e.g. the antibody specific for CD38 and lenalidomide, may beadministered together, or separately. When administered together, thetwo components may be formulated together in one pharmaceuticalcomposition, which may include a pharmaceutical acceptable carrier orexcipient. Alternatively the two components might also be formulated indifferent pharmaceutical compositions. In this case the two componentscan be administered simultaneously or subsequently. In an embodiment,the thalidomide or an analog thereof, e.g. lenalidomide, is administeredprior to and/or separately from the administration of the antibodyspecific for CD38, e.g. MOR202. In a further embodiment, lenalidomide,is administered at least 72 hours prior to administration of theantibody specific for CD38, e.g. MOR202. This time period allows forlenalidomide mediated upregulation of CD38 in the target cells.

A pharmaceutical composition includes an active agent, eg. an antibodyfor therapeutic use in humans. A pharmaceutical composition may includeacceptable carriers or excipients.

“Administered” or “administration” includes but is not limited todelivery by an injectable form, such as, for example, an intravenous,intramuscular, intradermal or subcutaneous route or mucosal route, forexample, as a nasal spray or aerosol for inhalation or as an ingestablesolution, capsule or tablet.

A “therapeutically effective amount” of a compound or combination refersto an amount sufficient to cure, alleviate or partially arrest theclinical manifestations of a given disease or disorder and itscomplications. The amount that is effective for a particular therapeuticpurpose will depend on the severity of the disease or injury as well ason the weight and general state of the subject. It will be understoodthat determination of an appropriate dosage may be achieved, usingroutine experimentation, by constructing a matrix of values and testingdifferent points in the matrix, all of which is within the ordinaryskills of a trained physician or clinical scientist.

Surprisingly, it was found that the combination of a particularanti-CD38 antibody and lenalidomide mediated a synergistic level ofAntibody-Dependent Cell-Mediated Cytotoxicity (ADCC) in both AMO-1 andNCI-H929 multiple myeloma cells. In addition, and also unexpectedly, aparticular anti-CD38 antibody when combined with lenalidomide mediated asynergistic level of reduction in bone lysis in the NCI-H929 SCID mousemodel and synergistically increased the median survival days in theRAMOS SCID mouse model. Therefore, the combination of the exemplifiedantibody specific for CD38 and lenalidomide behaved synergistically inboth the in vitro and in vivo models relevant to multiple myeloma and/ornon-Hodgkin's lymphoma. Therefore, this combination yields synergisticresults in the treatment of multiple myeloma and/or non-Hodgkin'slymphoma in humans.

Lenalidomide is a thalidomide analog, therefore, it is expected thatother thalidomide analogs, such as, pomalidomide or thalidomide itselfalso lead to synergistic effects when used in combination with ananti-CD38 antibody. In addition, as thalidomide or an analog thereofupregulate CD38 expression in multiple myeloma cell lines, therefore, itis expected that synergism should result when other agents thatupregulate the expression of CD38 on the surface of tumor cells, e.g.trans-retinoic acid, and anti-CD₃₈ antibodies are used in combination.

Surprisingly, it was found that the combination of a particularanti-CD38 antibody and bortezomib mediated a high level ofAntibody-Dependent Cell-Mediated Cytotoxicity (ADCC) in the NCI-H929 andLP-1 multiple myeloma cell lines. In addition, and also surprisingly itwas found that the combination of a particular anti-CD38 antibody andbortezomib mediated a synergistic level of reduction in bone lysis inthe NCI-H929 SCID mouse model and synergistically increased the mediansurvival days in the RAMOS SCID mouse model. Therefore, the combinationof the exemplified antibody specific for CD38 and bortezomib behavedsynergistically in the in vivo models relevant to multiple myelomaand/or non-Hodgkin's lymphoma. Therefore, this combination yieldssynergistic results in the treatment of multiple myeloma and/ornon-Hodgkin's lymphoma in humans.

It is expected that other proteasome inhibitors, such as, Disulfuram,Epigallocatechin-3-gallate, and Salinosporamide A will lead to similareffects when used in combination with an anti-CD38 antibody.

The “CDRs” herein are defined by either Chothia et al., Kabat et al. orby an internal numbering convention. See Chothia C, Lesk A M. (1987)Canonical structures for the hypervariable regions of immunoglobulins. JMol Biol., 196(4):901-17, which is incorporated by reference in itsentirety. See Kabat E. A, Wu T. T., Perry H. M., Gottesman K. S. andFoeller C. (1991). Sequences of Proteins of Immunological Interest. 5thedit., NIH Publication no. 91-3242, US Dept. of Health and HumanServices, Washington, D.C. which is incorporated by reference in itsentirety.

EMBODIMENTS

An aspect of the present disclosure comprises a synergistic combinationof an antibody specific for CD38 and (a) thalidomide or an analogthereof, or (b) a proteasome inhibitor, for use in the treatment ofmultiple myeloma and/or non-hodgkins lymphoma.

An aspect of the present disclosure comprises a combination of anantibody specific for CD38 and thalidomide or an analog thereof. Inembodiments, the combination is synergistic.

In embodiments, the antibody specific for CD38 comprises an HCDR1 regionof sequence GFTFSSYYMN (SEQ ID NO: 1) or of sequence SYYMN (SEQ ID NO:14), an HCDR2 region of sequence GISGDPSNTYYADSVKG (SEQ ID NO: 2), anHCDR3 region of sequence DLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region ofsequence SGDNLRHYYVY (SEQ ID NO: 4), an LCDR2 region of sequence GDSKRPS(SEQ ID NO: 5), and an LCDR3 region of sequence QTYTGGASL (SEQ ID NO:6).

In embodiments, the antibody specific for 0038 comprises an HCDR1 regionof sequence DYWMQ (SEQ ID NO: 15), an HCDR2 region of sequenceTIYPGDGDTGYAQKFQG (SEQ ID NO: 16), an HCDR3 region of sequenceGDYYGSNSLDY (SEQ ID NO: 17), an LCDR1 region of sequence KASQDVSTVVA(SEQ ID NO: 18), an LCDR2 region of sequence SASYRYI (SEQ ID NO: 19),and an LCDR3 region of sequence QQHYSPPYT (SEQ ID NO: 20).

In an aspect the combination is used for the treatment of multiplemyeloma and/or non-hodgkins lymphoma. Embodiments comprise acombination, wherein the thalidomide analog is lenalidomide.

An aspect relates to pharmaceutical compositions comprising thecombinations. In embodiments, the composition comprises an acceptablecarrier. In embodiments, the composition is administered in an effectiveamount.

An aspect of the present disclosure comprises a synergistic combinationof an antibody specific for CD38 comprising an HCDR1 region of sequenceGFTFSSYYMN (SEQ ID NO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2region of sequence GISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region ofsequence DLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequenceSGDNLRHYYVY (SEQ ID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ IDNO: 5), and an LCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) andlenalidomide for the treatment of multiple myeloma and/or non-hodgkinslymphoma.

A further embodiment comprises a combination, wherein the antibodycomprises a variable heavy chain of the sequenceQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTV SS (SEQ IDNO: 10) and a variable light chain of the sequenceDIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVLGQ (SEQ ID NO: 11).

An aspect of the present disclosure comprises a synergistic combinationof an antibody specific for CD38 comprising an HCDR1 region of sequenceDYWMQ (SEQ ID NO: 15), an HCDR2 region of sequence TIYPGDGDTGYAQKFQG(SEQ ID NO: 16), an HCDR3 region of sequence GDYYGSNSLDY (SEQ ID NO:17), an LCDR1 region of sequence KASQDVSTVVA (SEQ ID NO: 18), an LCDR2region of sequence SASYRYI (SEQ ID NO: 19), and an LCDR3 region ofsequence QQHYSPPYT (SEQ ID NO: 20) and lenalidomide for the treatment ofmultiple myeloma and/or non-hodgkins lymphoma.

A further embodiment comprises a combination, wherein the antibodycomprises a variable heavy chain of the sequenceQVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGTIYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDYYGSNSLDYWGQGTSV TVSS (SEQ IDNO: 21) and a variable light chain of the sequenceDIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGGTKLEIKRT (SEQ ID NO: 22).

In embodiments the antibody has an IgG1 Fc region. In embodiments theantibody comprises a modified Fc region, wherein said modificationenhances ADCC activity.

In another aspect, the components of the combination, the antibodyspecific for CD38 and lenalidomide, are administered separately. In anembodiment, lenalidomide is administered prior to administration of theantibody specific for CD38. In a further embodiment, lenalidomide isadministered at least 72 hours prior to administration of the antibodyspecific for CD38.

In another aspect the synergistic combination of an antibody specificfor CD38 comprising an HCDR1 region of sequence GFTFSSYYMN (SEQ IDNO: 1) or of sequence SYYMN (SEC ID NO: 14), an HCDR2 region of sequenceGISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region of sequenceDLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequence SGDNLRHYYVY (SEQID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ ID NO: 5), and anLCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) and lenalidomide isable to mediate killing of CD38-expressing AMO-1 cells and/or NCI-H929cells by ADCC in the presence of isolated human PBMCs with an at leasttwo-fold, three-fold, four-fold, or five-fold better efficacy thanlenalidomide alone.

In another aspect the synergistic combination of an antibody specificfor CD38 comprising an HCDR1 region of sequence GFTFSSYYMN (SEQ IDNO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2 region of sequenceGISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region of sequenceDLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequence SGDNLRHYYVY (SEQID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ ID NO: 5), and anLCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) and lenalidomide isable to reduce bone lysis with an at least two-fold, three-fold,four-fold, or five-fold better efficacy than lenalidomide alone.

Another aspect comprises a method of treating multiple myeloma and/ornon-hodgkins lymphoma in an individual in need thereof, which methodcomprises administration of an antibody specific for CD38 comprising anHCDR1 region of sequence GFTFSSYYMN (SEQ ID NO: 1) or of sequence SYYMN(SEQ ID NO: 14), an HCDR2 region of sequence GISGDPSNTYYADSVKG (SEQ IDNO: 2), an HCDR3 region of sequence DLPLVYTGFAY (SEQ ID NO: 3), an LCDR1region of sequence SGDNLRHYYVY (SEQ ID NO: 4), an LCDR2 region ofsequence GDSKRPS (SEQ ID NO: 5), and an LCDR3 region of sequenceQTYTGGASL (SEQ ID NO: 6) and lenalidomide to an individual havingmultiple myeloma or non-hodgkins lymphoma. In embodiments, thecombination is administered in an effective amount.

Another aspect comprises a combination comprising an antibody specificfor CD38 comprising an HCDR1 region of sequence GFTFSSYYMN (SEQ IDNO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2 region of sequenceGISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region of sequenceDLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequence SGDNLRHYYVY (SEQID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ ID NO: 5), and anLCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) and lenalidomide. Inan embodiment, the combination is used for the treatment of cancer. In afurther embodiment, the cancer is selected from multiple myeloma, andnon-hodgkins lymphoma.

Another aspect comprises a combination of an antibody specific for CD38and a proteasome inhibitor. In embodiments, the combination issynergistic. In embodiments, the antibody specific for CD38 comprises anHCDR1 region of sequence GFTFSSYYMN (SEQ ID NO: 1) or of sequence SYYMN(SEQ ID NO: 14), an HCDR2 region of sequence GISGDPSNTYYADSVKG (SEQ IDNO: 2), an HCDR3 region of sequence DLPLVYTGFAY (SEQ ID NO: 3), an LCDR1region of sequence SGDNLRHYYVY (SEQ ID NO: 4), an LCDR2 region ofsequence GDSKRPS (SEQ ID NO: 5), and an LCDR3 region of sequenceQTYTGGASL (SEQ ID NO: 6).

In an aspect the combination is used for the treatment of multiplemyeloma and/or non-hodgkins lymphoma. In embodiments, the combinationcomprises a proteasome inhibitor, which is bortezomib. An aspect relatesto pharmaceutical compositions comprising the combinations. Inembodiments, the composition comprises an acceptable carrier. Inembodiments, the composition is administered in an effective amount.

An aspect of the present disclosure comprises a synergistic combinationof an antibody specific for CD38 comprising an HCDR1 region of sequenceGFTFSSYYMN (SEQ ID NO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2region of sequence GISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region ofsequence DLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequenceSGDNLRHYYVY (SEQ ID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ IDNO: 5), and an LCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) andbortezomib for the treatment of multiple myeloma and/or non-hodgkinslymphoma.

A further embodiment comprises a combination, wherein the antibodycomprises a variable heavy chain of the sequenceQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTV SS (SEQ IDNO: 10) and a variable light chain of the sequenceDIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVLGQ (SEQ ID NO: 11).

In embodiments the antibody has an IgG1 Fc region. In embodiments theantibody comprises a modified Fc region, wherein said modificationenhances ADCC activity.

In an embodiment, the combination is used for the treatment of cancer.In a further embodiment, the cancer is selected from multiple myeloma,and non-hodgkins lymphoma.

In another aspect, the components of the combination, the antibody andproteasome inhibitor, are administered separately.

In another aspect the synergistic combination of an antibody specificfor CD38 comprising an HCDR1 region of sequence GFTFSSYYMN (SEQ IDNO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2 region of sequenceGISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region of sequenceDLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequence SGDNLRHYYVY (SEQID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ ID NO: 5), and anLCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) and bortezomib is ableto mediate killing of CD38-expressing LP-1 cells and/or NCI-H929 cellsby ADCC in the presence of isolated human PBMCs with an at leasttwo-fold, three-fold, fourfold, or five-fold better efficacy thanbortezomib alone.

In another aspect the synergistic combination of an antibody specificfor CD38 comprising an HCDR1 region of sequence GFTFSSYYMN (SEQ IDNO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2 region of sequenceGISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region of sequenceDLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequence SGDNLRHYYVY (SEQID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ ID NO: 5), and anLCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) and bortezomib is ableto reduce bone lysis with an at least two-fold, three-fold, four-fold,or five-fold better efficacy than bortezomib alone.

In another aspect, the present disclosure comprises a method of treatingmultiple myeloma and/or non-hodgkins lymphoma in an individual in needthereof, which method comprises administration of an antibody specificfor CD38 comprising an HCDR1 region of sequence GFTFSSYYMN (SEQ IDNO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2 region of sequenceGISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region of sequenceDLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequence SGDNLRHYYVY (SEQID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ ID NO: 5), and anLCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) and bortezomib to anindividual having multiple myeloma or non-hodgkins lymphoma.

In embodiments, the combination is administered in an effective amount.

Another aspect comprises a combination comprising an antibody specificfor CD38 comprising an HCDR1 region of sequence GFTFSSYYMN (SEQ IDNO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2 region of sequenceGISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region of sequenceDLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequence SGDNLRHYYVY (SEQID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ ID NO: 5), and anLCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) and bortezomib.

An aspect comprises a synergistic combination of an antibody specificfor CD38 comprising an HCDR1 region of sequence GFTFSSYYMN (SEQ IDNO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2 region of sequenceGISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 of sequence DLPLVYTGFAY (SEQID NO: 3), an LCDR1 region of sequence SGDNLRHYYVY (SEQ ID NO: 4), anLCDR2 region of sequence GDSKRPS (SEQ ID NO: 5), and an LCDR3 region ofsequence QTYTGGASL (SEQ ID NO: 6) and

(a) thalidomide or an analog thereof, or

(b) a proteasome inhibitor,

for use in the treatment of multiple myeloma and/or non-hodgkinslymphoma.

Embodiments comprise a combination, wherein the antibody comprises avariable heavy chain of the sequenceQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTV SS (SEQ IDNO: 10) and a variable light chain of the sequenceDIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVLGQ (SEQ ID NO: 11).

Embodiments comprise a combination, wherein the antibody comprises anIgG1 Fc region. Embodiments comprise a combination, wherein the antibodycomprises a modified Fc region, wherein said modification enhances ADCCactivity.

Embodiments comprise a combination, wherein said antibody specific forCD38 and said thalidomide or an analog thereof or proteasome inhibitorare administered separately.

Embodiments comprise a combination, which is able to reduce bone lysiswith an at least two-fold better efficacy than lenalidomide and/orbortezomib alone.

Embodiments comprise a combination, wherein said antibody specific forCD38 is combined with thalidomide or an analog thereof. Embodimentscomprise a combination, wherein the thalidomide analog compriseslenalidomide. Embodiments comprise a combination, wherein lenalidomideis administered prior to administration of the antibody specific forCD38. Embodiments comprise a combination, wherein lenalidomide isadministered at least 72 hours prior to administration of the antibodyspecific for CD38.

Embodiments comprise a combination of an antibody specific for CD38 andlenalidomide, which is able to mediate killing of CD38-expressing AMO-1and/or NCI-H929 cells by ADCC in the presence of isolated human PBMCswith an at least two-fold better efficacy than lenalidomide alone.

Embodiments comprise a combination, comprising said antibody specificfor CD38 and a proteasome inhibitor. In some embodiments, the proteasomeinhibitor is bortezomib. Embodiments comprise a combination of anantibody specific for CD38 and bortezomib, which is able to mediatekilling of CD38-expressing LP-1 and/or NCI-H929 cells by ADCC in thepresence of isolated human PBMCs with an at least two-fold betterefficacy than bortezomib alone.

An aspect comprises a synergistic combination of an antibody specificfor CD38 comprising an HCDR1 region of sequence GFTFSSYYMN (SEQ IDNO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2 region of sequenceGISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region of sequenceDLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequence SGDNLRHYYVY (SEQID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ ID NO: 5), and anLCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) and lenalidomide orother thalidomide analog for use in the treatment of multiple myelomaand/or non-hodgkins lymphoma.

An aspect comprises a synergistic combination of an antibody specificfor CD38 comprising an HCDR1 region of sequence GFTFSSYYMN (SEQ IDNO: 1) or of sequence SYYMN (SEQ ID NO: 14), an HCDR2 region of sequenceGISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region of sequenceDLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequence SGDNLRHYYVY (SEQID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ ID NO: 5), and anLCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6) and bortezomib orother proteasome inhibitor for use in the treatment of multiple myelomaand/or non-hodgkins lymphoma.

EXAMPLES Example 1 CD38 Expression on the Surface of Various Cell Lines

The cell lines of Table 1 were tested for levels of CD38 expression.

TABLE 1 Cell Line Supplied by: Cultivated in: AMO-1: Multiple DSMZ #ACC538 RPMI1640, with Myeloma Cell Line L-Glutamine, (PAN Biotech GmbH, CatNo.: P04-16500 medium) LP1: Multiple DSMZ #ACC 41 Iscove's ModifiedDulbecco's Myeloma Cell Line Medium (IMDM) with GlutaMAX ™ (Invitrogen,Cat No.: 31980-048) NCI-H929: Multiple DSMZ #ACC 163 RPMI1640 (same asMyeloma Cell Line AMO-1), supplemented with 1 mM Na-Pyruvate, 50 μMβ-Mercaptoethanol RPM18226: Multiple DSMZ #ACC 402 RPMI1640 (same asAMO-1) Myeloma Cell Line OPM-2: Multiple DSMZ #ACC 50 RPMI1640 (same asAMO-1) Myeloma Cell Line Plasmacytoma, Klinikum rechts RPMI1640 (same asAMO-1) Malignant Plasma Cells der lsar

Bone marrow samples (4-10 ml aspirate) from multiple myeloma patientsand extramedullary tumor plasmacytoma samples were obtained afterinformed consent from the Klinikum rechts der Isar (“KrdI”) (Munich,Germany). Samples were subjected to centrifugation, and further plasmacell enrichment was achieved via magnetic-activated cell sorting.

Cells were stained with a directly labelled QuantiBRITE™ CD38-PEantibody (Becton Dickinson GmbH, Clone HB7, CAT #342371), which isspecific for CD38. The “Antibodies Bound Per Cell” (ABC's) weredetermined using the flow cytometry based QuantiBRITE™ system, whichmeasures the geometric mean (GeoMean) per cell. Conversion of measuredGeoMean into correlating ABC amount per cell was done with GraphPadPRISM™ software. The ABC values are assumed to correlate with the numberof CD38 molecules per cell, since QuantiBRITE™ CD38-PE carries one PEmolecule per antibody. The results are shown in Table 2.

Example 2 Evaluation of Effect of Lenalidomide on Upregulation of CD38in Various Cell Lines

To determine whether lenalidomide induced upregulation of CD38 in themultiple myeloma and plasmacytoma cells of Table 1, the cell lines wereincubated with 100 μM lenalidomide and, subsequently, CD38 surfaceexpression was analyzed by FACS.

Materials and Methods

Around 2×10⁵ cells of each of the cells lines of Table 1 were plated on48-well dishes in standard RPMI medium. Lenalidomide, purchased fromSelleck Chemicals (LLC S1029, CAS No. 191732-6; Batch: S10290), wasapplied to respective wells to a final concentration of 100 μM in avolume of 750 μl containing 20% FCS and 0.1% DMSO. As negative control0.1% DMSO in FCS-supplemented medium was used and plates were incubatedfor 24 h, 48 h and 72 h at 37° C. and 5% CO₂ in humidified incubator.

Cells were resuspended by gentle pipetting and 250 μl of cell suspensionper incubation period were transferred into a well of a 96-well roundbottom plate. Cells were washed by centrifugation for 1 min at 700×g andwere resuspended in 150 μl of cold FACS buffer (1×PBS supplemented with3% FCS). Cells were again pelleted down by centrifugation and wereresuspended in 150 μl of FACS buffer containing 15 μg/ml of anti-CD38antibody (MOR202, IgG1) or control antibody MOR03207 and incubated for 1h on ice. Cells were then washed 3 times by centrifugation and wereresuspended in FACS buffer supplemented with PE-labeled secondaryantibody (PE-Fab₂ fragment, goat anti-human IgG. Fc-fragment specific;Jackson Immuno Research; CAT: 109-116-098; Lot: 80938). Cells wereincubated for 45 minutes on ice, then washed 3 times by centrifugationand resuspended in FACS buffer. The cell suspensions were then subjectedto FACS analysis using a FACS array device.

The basal CD38 expression of each cell line and the affect oflenalidomide on CD38 expression are shown in Table 2. Additionally, theaffect of lenalidomide on the CD38 expression of AMO-1 cells is shown inFIG. 1, and the affect of lenalidomide on the CD38 expression ofNCI-H929 cells is shown in FIG. 5.

TABLE 2 Absolute number of ABC (CD38 expression) LEN (extrap- Fold Cellline Basal olated) INCREASE increase Effect AMO-1 25,000 115,000 90,0004.5 Significant LP-1 125,000 162,500 37,500 1.3 No NCI-H929 195,000390,000 195,000 2.0 Weak RPMI-8226 670,000 871,000 199,000 1.3 WeakOPM-2 38,000 98,800 60,800 2.6 Significant Plasma- 30,000 69,000 39,0002.3 Significant cytoma

Example 3 Inhibition of Proliferation of AMO-1 Cells Using LenalidomideAlone

The cytotoxicity of Lenalidomide was tested in AMO-1 cells. Cells werecollected and distributed in 96-well plates with 5000 cells per well.Increasing amounts of Lenalidomide were added to the wells and plateswere incubated for 24 h, 48 h and 72 h at 37° C. in a humidifiedincubator (5% CO₂).

After incubation, plates were analyzed for cell proliferation in aquantitative colorimietric XTT-based assay using the cell proliferationkit II (ROCHE, Cell Proliferation Kit II, Cat. No.: 11465015001). Forsubsequent measurement plates were subjected to Tecan Genios Reader andabsorbance at 492 nm was detected.

The results are shown in FIG. 2.

Example 4 Synergistic Combination of MOR0202 and Lenalidomide in AMO-1Cells

AMO-1 cells were selected for testing with the combination of MOR202 andlenalidomide. AMO-1 cells are similar to plasmacytoma cells in humans inthat both have a low basal CD38 expression, and CD38 is significantlyupregulated in both upon treatment with lenalidomide as shown in Table2.

PBMC's were isolated by density gradient centrifugation of freshlyisolated human blood. Isolated blood from different donors were layeredon a defined volume of Biocoll (Biochrome AG; CAT No.:L6115; LOTNo.:1050T) in a Falcon tube and centrifuged at 380 g. The PBMCs wereisolated and supplemented with RPMI medium.

After 72 h, cells were counted and the PBMCs were adjusted to aconcentration of 6.6×106/ml while the AMO-1 cells were adjusted to afinal concentration of 2.5×10⁵/ml. For later identification in flowcytometry, the AMO-1 cells were were stained for 3 min with 0.1 μg/ml ofCalceinAM (Calcein: 1 mg/ml stock solution, Invitrogen, Cat No.: C3099)and washed three times by gentle centrifugation. 100 μl of target cellsuspension were mixed with 100 μl of PBMCs to achieve a ratio of 1:30.Antibody MOR202 or antibody MOR03207 (negative control) were added to afinal concentration of 15 μg/ml. Cell suspensions were further incubatedfor 4 h at 37° C. To detect dead AMO-1 cells, cell suspensions werechallenged with propidium iodide (PI) and subsequently analyzed in flowcytometry. Target cells were separated via gating of CalceinAM positivecell populations, and cells killed via ADCC were quantified.

In total six experiments were performed in order to determine themediation of ADCC on AMO-1 cells by the combination of MOR202 andlenalidomide. In three experiments, the PBMCs and AMO-1 cells weretreated with lenalidomide prior to treatment with MOR202, the resultsare shown in Tables 3 a-c and FIG. 3. In three additional experiments,only the PBMCs were treated with lenalidomide prior to treatment withMOR202, the results are shown in Tables 4 a-c and FIG. 4.

Table 3 Both Effector and AMO-1 cells were treated with Lenalidomideprior to treatment with MOR202. Single and combination doses of 10 μMLEN and 15 μg/m of MOR03207 and MOR202 were used.

The data is presented in the following three ways, as a) raw data (%dead cells), b) normalized specific killing data, where the MOR202treatment group is set as 1 (100%), and c) normalized specific killingdata, where the theoretical combination is set as 1 (100%). Table 3arepresents raw data.

TABLE 3a Combination LEN of LEN (10 μM) DMSO LEN MOR202 (10 μM) MOR03207alone control 10 μM alone and MOR202 MOR03207 (15 μg/ml) + LEN withoutwithout AMO-1 alone (15 μg/ml) (15 μg/ml) DMSO (15 μg/ml) DMSO (0 μM)PBMCs PBMCs Exp. 1 12.89 23.69 35.98 13.10 14.15 15.12 15.45 15.41 11.07Exp. 2 10.13 22.53 29.09 7.94 10.52 6.99 13.22 8.44 8.45 Exp. 3 22.8049.56 80.39 19.93 24.04 22.24 22.63 22.38 26.43

The units of the values listed are % dead cells. The DMSO, MOR03207,MOR03207+DMSO, LEN0, LEN10 without PBMCs and DMSO without PBMCs groupsare controls.

Table 3b represents the data of Table 3a, but normalized, where theMOR202 treatment group is set as 1 (100%).

TABLE 3b MOR202 (15 μg/ml) MOR03207 MOR202 LEN alone Theoretical and LENAMO-1 (15 μg/ml) (15 μg/ml) (10 μM) combination (10 μM) Exp. 1 −0.1 1.00.0 1.0 2.2 Exp. 2 −0.2 1.0 0.2 1.2 1.8 Exp. 3 0.1 1.0 0.1 1.1 2.3

For Tables 3b-c, “Theoretical Combination” represents the addition ofthe values of MOR202 alone and the values of LEN alone. The normalizeddata of Table 3b is calculated as follows. Table 3a represents thenumber of dead cells. Therefore, the specific killing values of Table 3bare calculated by subtracting the values of the controls. Then thespecific killing values are compared to the MOR202 group, which is setas 1. The averages of the results in Table 3b are shown in FIG. 3.

1. Determination of Synergism

1.1 Chou et al.

The methods of Chou-Talalay were used to determine synergism. See Chou TC, Talalay P, Quantitative analysis of dose-effect relationships: thecombined effects of multiple drugs or enzyme inhibitors. Adv EnzymeRegul 22: 27-55 (1984), which is incorporated by reference in itsentirety. Synergism analysis is carried out using the CI-isobol method.

Median-Effect Equation

The median-effect equation models of the effect of an inhibitor (such asa drug) asF _(a) /F _(u)=(D/D50)^mwhere D is the dose, F_(a) and F_(u) is the fraction of the systemaffected and unaffected by the dose D (F_(a)+F_(u)=1); D50 is the doseproducing the median effect (e.g. IC50, ED50, LD50). The constant mdetermines the shape of the dose-effect curve.We used Excel Fit software to carry out a linear regression calculationto estimate the parameters m and D50.

The effects of the combination on AMO-1 cells is measured % cell deathas described above. We define the fraction F_(u) to be the ratio of %cell death of the treated cell line to the °/° cell death of the cellline exposed to a control. That is:F _(u)=% cell death(treated cell line)/% cell death(non-treated cellline)

Then the % cell death of a cell line is the constant D50 in the medianeffect equation, which can be estimated by the linear regressiondescribed above.

CI-isobol Method

The CI-isobol method provides a quantitative assessment of synergismbetween drugs. A combination index (CI) is estimated from dose-effectdata of single and combined drug treatments. A value of CI less than 1indicates synergism; CI=1 indicates additive effect; and CI>1 indicatesantagonism. Synergistic ranges are further defined by Chou and Talahayfor CI values<0.1 as very strong synergism. CI values between 0.1 and0.3 as strong synergism. CI values of 0.3-0.7 as synergism, CI values of0.7-0.9 as moderate to slight synergism. Drug interaction (synergism orantagonism) is more pronounced the farther a CI value is from 1.

Formally, the combination index (CI) of a combined drug treatment isdefined asCI=D ₁ /D _(x1) +D ₂ /D _(x2)

Here D1 and D2 are the doses of drug 1 and drug 2, respectively, in thecombination; Dx1, and Dx2 each is the dose of a treatment with only drug1 and drug 2 that would give the same effect as that of the combination,respectively. The doses Dx1 and Dx2 need to be estimated from thedose-effect data of single drug treatments. Essentially, a median effectequation is fitted to the data of each drug. From the median effectequation of a drug, we can estimate the dose (i.e. D) necessary toproduce an effect (i.e. Fa, Fu). The further a point lies from theadditive line, the bigger the different between 1 and its CI, thus thestronger the (synergistic or antagonistic) effect is.

The above method is described in Chou T C, Talalay P, Quantitativeanalysis of dose-effect relationships: the combined effects of multipledrugs or enzyme inhibitors. Adv Enzyme Regul 22: 27-55 (1984), which isincorporated by reference in its entirety. An additional review of theabove Chou method is also provided in Ting-Chao Chou, Theoretical Basis,Experimental Design, and Computerized Simulation of Synergism andAntagonism in Drug Combination Studies, Pharmacol Rev 58:621-681 (2006),which is incorporated by reference in its entirety.

The curves generated for the Chou based synergy calculations are shownin FIGS. 12-18. In FIG. 12, the best fit curve was determined byremoving the data points a) where the concentration of MOR202 was toolow to have any effect and b) where the concentration was nearsaturation. At the appropriate date point, approx. 80% cell killing, theCI value is less than 1, supporting clear synergy. FIGS. 13-18 representthe six experiments from Tables 3 and 4, and in each the Dx1 (dose ofMOR202) needed to reach 100% effect of the combination of MOR 202 andlenalidomide goes to infinity; therefore, the D₁/D_(x1) is less than 1and as lenalidomide has no effect on AMO-1 cells regarding cell killing,the Dx2 value also approaches infinity, so the D₂/D_(x2) approximates 0,therefore the CI values of each of the six experiments is less than 1,supporting clear synergy.

Table 3c represents the normalization of data, where the theoreticalcombination is set as 1 (100%) and includes the CI Chou calculations.

TABLE 3c MOR202 (0.42 μg/ml) MOR202 LEN Theoretical and LEN CombinationCell line Experiment (0.42 μg/ml) (5 μM) Combination (5 μM) Index (CI)Conclusion AMO-1 Experiment 0.6 0.4 1.0 1.0 <<0.1 * synergy 1 Experiment0.9 0.1 1.0 2.4 <<0.1 * synergy 2 Experiment 0.9 0.1 1.0 1.8 <<0.1 *synergy 3 AVERAGE 0.8 0.2 1.0 1.7 — —

The date shown in Table 3c differs from Tables 3a and 3b. Table 3c isbased upon different raw data points than shown in Table 3a, as theconcentrations chosen in Table 3c are closer to the EC₅₀ of the antibody(raw data not shown). “Theoretical Combination” represents the additionof the values of MOR202 alone and the values of LEN alone.

Table 4 Effector cells only treated with Lenalidomide prior to treatmentwith MOR202. Single and combination doses of 10 μM LEN and 15 μg/ml ofMOR03207 and MOR202 were used.

The data is presented in the following three ways, as a) raw data (%dead cells), b) normalized specific killing data, where the MOR202treatment group is set as 1 (100%), and c) normalized specific killingdata, where the theoretical combination is set as 1 (100%). Table 4arepresents raw data.

TABLE 4a Combination of LEN LEN MOR202 (10 μM) MOR03207 10 μM alone andMOR202 MOR03207 15 μg/ml + LEN AMO-1 alone (15 μg/ml) (15 μg/ml) DMSO 15μg/ml DMSO (0 μM) Exp. 1 15.33 23.09 23.46 14.62 16.17 15.97 12.87 Exp.2 12.98 21.08 25.75 10.24 12.17 11.45  9.78 Exp. 3 17.93 48.28 56.4916.75 17.42 15.77 18.16

The units of the values listed are % dead cells. The DMSO, MOR03207.MOR03207+DMSO, LEN0, LEN10 without PBMCs and DMSO without PBMCs arecontrols.

TABLE 4b MOR202 (15 μg/ml) MOR03207 MOR202 LEN alone Theoretical and LENAMO-1 (15 μg/ml) (15 μg/ml) (10 μM) combination (10 μM) Exp. 1 0.5 1.00.1 1.1 1.1 Exp. 2 0.3 1.0 0.3 1.3 1.6 Exp. 3 0.0 1.0 0.0 1.0 1.3

Table 4b represents the data of Table 4a, but normalized, where theMOR202 treatment group is set as 1 (100%). For Tables 4b-c, “Theoreticalcombination” represents the values of MOR202 alone plus the values ofLEN alone.

The normalization of the data as shown in Table 4b is calculated asdescribed in Table 3b, by substracting the controls. The averages of theresults of Table 4b are shown in FIG. 4.

TABLE 4c MOR202 (0.42 μg/ml) MOR202 LEN Theoretical and LEN CombinationCell line Experiment (0.42 μg/ml) (5 μM) Combination (5 μM) Index (CI)Conclusion AMO-1 Experiment 1.2 −0.2 1.0 1.7 <<0.1 * synergy 1Experiment 0.7 0.3 1.0 1.4 <<0.1 * synergy 2 Experiment 0.8 0.2 1.0 1.3<<0.1 * synergy 3 AVERAGE 0.9 0.1 1.0 1.5 — —

Table 4c represents the normalization of the data, where the theoreticalcombination is set as 1 (100%) and includes the CI Chou et al.calculations using the methodology described above within Example 4.

Table 4c differs from Tables 4a and 4b. Table 4c is based upon differentraw data points than shown in Table 4a, as the concentrations chosen inTable 4c are closer to the EC₅₀ of the antibody (raw data not shown),

1. Determination of Synergism

1.2 Clarke et al. Synergism

Where one drug has low activity, as in here where Lenalidomide alone haslow cytotoxity against AMO-1 cells, synergy can also be determined bystatistical evidence that the combination is significantly differentfrom the inhibitory drug alone. See Clarke et al., Issues inexperimental design and endpoint analysis in the study of experimentalcytotoxic agents in vivo in breast cancer and other models. BreastCancer Research and Treatment 46:255-278 (1997), which is incorporatedby reference in its entirety. Here both Chou et al. as shown above andthe methods of Clarke et al. were used in the determination ofsynergism.

The data is analysed in the following way:Antagonistic(AB)/C<(A/C)×(B/C)Additive(AB)/C=(A/C)×(B/C)Synergistic(AB)/C>(A/C)×(B/C)where A is the treatment with LEN alone; B is the treatment with MOR202alone; C is response to the treatment vehicle; AB is combination oftreatments A and B.

TABLE 5 The raw data values shown in this table are the same as thoseshown in Table 3a, as they come from the same three experiments, whereboth effector and AMO-1 cells were treated with Lenalidomide prior totreatment with MOR202 and the single and combination doses of 10 μM LENand 15 μg/ml of MOR03207 and MOR202 were used. The only difference isthat the data is analyzed using Clarke et al. instead of Chou et al.Experiment 1 Experiment 2 Experiment 3 A: LEN alone 15.41 8.44 22.38 B:MOR202 alone 23.69 22.53 49.56 C: control 11.07 8.45 26.43 AB:combination of 5.98 29.09 80.39 LEN and MOR202 (AB)/C 3.25 3.44 3.04(A/C) × (B/C) 2.98 2.66 1.59  A = response to treatment with LEN alone B = response to treatment with MOR202 alone  C = response to treatmentwith control AB = combination of treatments A and B The values of A, B,C and AB represent % cell killing.

In each experiment (AB)/C is greater than (A/C)×(B/C), showing clearsynergy.

TABLE 6 The raw data values shown in this table are the same as thoseshown in Table 4a, as they come from the same three experiments, whereonly the effector cells were treated with Lenalidomide prior totreatment with MOR202 and the single and combination doses of 10 μM LENand 15 μg/ml of MOR03207 and MOR202 were used. The only difference isthat the data is analyzed using Clarke et al. instead of Chou et al.Experiment 1 Experiment 2 Experiment 3 A: LEN alone 15.33 12.98 17.93 B:MOR202 alone 23.09 21.08 48.28 C: Control 15.97 11.45 15.77 AB:combination of 23.46 25.75 56.49 LEN and MOR202 (AB)/C 1.47 2.25 3.58(A/C) × (B/C) 1.39 2.09 3.48  A = response to treatment with LEN alone B = response to treatment with MOR202 alone  C = response to treatmentwith control AB = combination of treatments A and B

In each experiment (AB)/C is greater than (A/C)×(B/C), showing clearsynergy.

Results

Applying the analysis of Clarke et al., LEN synergistically enhancedMOR202 ADCC activity in AMO-1 cells in all 6 experiments. Applying theanalysis of Chou et al., LEN synergistically enhanced MOR202 ADCCactivity in AMO-1 cells in 6 out of 6 experiments. This enhancement ofactivity was identified to be by several mechanisms including directcytotoxicity, activation of effector cells and upregulation of CD38expression levels on MM cells.

Experiments according to example 4 are also performed with otherantibodies specific for CD38, for example, the “Ref mAB5” antibody,

Example 5 Inhibition of Proliferation of NCI-H929 Cells UsingLenalidomide Alone

The cytotoxicity of Lenalidomide was tested in NCI-H929 using themethods described in Example 3. The results are shown in FIG. 2. Insummary, challenge with Lenalidomide alone signficantly inhibited cellproliferation in NCI-H929 cells.

Example 6 Synergistic Combination of MOR202 and Lenalidomide in NCI-H929Cells

NCI-H929 cells were selected for testing with the combination of MOR202and lenalidomide. NCI-H929 cells express higher levels of CD38 thanAMO-1 cells, therefore, are representative of certain cells types foundin human patients with multiple myeloma or non-Hodgkin's lymphoma.

In total six experiments were performed, using the methods described inExample 4, in order to determine the mediation of ADCC on NCI-H929 cellsby the combination of MOR202 and lenalidomide. In three experiments, thePBMCs and NCI-H929 cells were treated with lenalidomide prior totreatment with MOR202, the results are shown in Tables 7a-b and FIG. 6.In three additional experiments only the PBMCs were treated withlenalidomide prior to treatment with MOR202, the results are shown inTables 8a-b and FIG. 7.

Table 7 Both Effector and NCI-H929 cells were treated with Lenalidomideprior to treatment with MOR202. Single and combination doses of 5 μM LENand 15 μg/ml of MOR03207 and 0.2 or 0.07 μg/ml MOR202 were used.

The data is presented in the following ways, as a) raw data (% deadcells), and b) normalized specific killing data, where the fractionalproduct combination is set as 1 (100%). Table 7a represents raw data.

TABLE 7a Combination of MOR202 LEN (5 μM) and LEN alone MOR202 MOR032075 μM (0.2* or (0.2* or LEN (15 μg/ml) + MOR03207 NCI-H929 alone 0.07μg/ml) 0.07 μg/ml) DMSO (0 μM) DMSO (15 μg/ml) Exp. 1 38.65 30.64*60.20* 18.01 18.42 18.27 17.81 Exp. 2 41.92 43.08 66.62 18.77 19.9220.26 19.20 Exp. 3 39.92 32.54 64.58 12.32 12.44 13.74 14.09

The units of the values listed are % dead cells. The DMSO, MOR03207,MOR03207+DMSO, LEN0, LEN10 without PBMCs and DMSO without PBMCs arecontrols.

Table 7b represents normalized data, where the fractional productcombination is set as 1 (100%).

TABLE 7b Combination Combination of MOR202 based upon LEN (5 μM) andalone LEN fractional MOR202 (0.2* or 5 μM product (0.2* or CombinationNCI-H929 0.07 μg/ml) alone concept 0.07 μg/ml) Index (CI) ConclusionExp. 1 0.42* 0.67 1.00 1.36* <<0.1 synergism Exp. 2 0.58 0.56 1.00 1.12<<0.1 synergism Exp. 3 0.45 0.67 1.00 1.24 <<0.1 synergism AVERAGE 0.480.63 1.00 1.24

The fractional product combination is calculated using the followingformula 1−[(1−A)*(1−B)]=fpc(%) as described in Ting-Chao Chou,Theoretical Basis, Experimental Design, and Computerized Simulation ofSynergism and Antagonism in Drug Combination Studies, Pharmacol Rev58:621-681 (2006), which is incorporated by reference in its entirety.Table 7b is based upon the raw data shown in Table 7a. The normalizationof the data as shown in Table 7b is calculated as described in Table 3b,by substracting the controls. In Table 7b, where the combination of LENand MOR202 is greater than the combination based upon the fractionalproduct concept, then clear synergy exists. In addition, CombinationIndex values were calculated using the methods of Chou et al. asdescribed in Example 4. The averages of the results of Table 7b areshown in FIG. 6.

Table 8 Effector cells only treated with Lenalidomide prior to treatmentwith MOR202. Single and combination doses of 5 μM LEN and 15 μg/ml ofMOR03207 and 0.2* or 0.07 μg/m MOR202 were used.

The data is presented in the following ways, as a) raw data (% deadcells), and b) normalized specific killing data, where the fractionalproduct combination is set as 1 (100%). Table 8a represents raw data.

TABLE 8a Combination of MOR202 LEN (5 μM) and LEN alone MOR202 MOR032075 μM (0.2* or (0.2* or LEN (15 μg/ml) + MOR03207 NCI-H929 alone 0.07μg/ml) 0.07 μg/ml) DMSO (0 μM) DMSO (15 μg/ml) Exp. 1 17.50 26.60*29.11* 18.36 17.56 19.52 17.07 Exp. 2 25.72 47.00 51.23 22.55 24.9024.16 23.19 Exp. 3 26.27 53.74 67.99 25.29 25.16 24.43 27.10

The units of the values listed are % dead cells. The DMSO, MOR03207,MOR03207+DMSO, LEN0, LEN10 without PBMCs and DMSO without PBMCs arecontrols.

Table 8b represents the normalized data, where the fractional productcombination is set as 1 (100%).

TABLE 8b Combination Combination of MOR202 based upon LEN (5 μM) andalone LEN fractional MOR202 (0.2* or 5 μM product (0.2* or CombinationNCI-H929 0.07 μg/ml) alone concept 0.07 μg/ml) Index (CI) ConclusionExp. 1 1.09* −0.10 1.00 1.10* 0.07 synergism Exp. 2 0.91 0.12 1.00 1.030.81 synergism Exp. 3 0.97 0.04 1.00 1.59 <<0.1 synergism AVERAGE 0.990.02 1.00 1.24

Table 8b is based upon the raw data shown in Table 8a. The normalizationof the data as shown in Table 8a is calculated as described in Table 3b,by substracting the controls. In Table 8b, where the combination of LENand MOR202 is greater than the combination based upon the fractionalproduct concept, then clear synergy exists. In addition, CombinationIndex values were calculated using the methods of Chou et al. asdescribed in Example 4. The averages of the results of Table 8b areshown in FIG. 7.

Determination of Synergism

1.3 Fractional Product Concept

The evaluation of the data in this example differs from that used in theanalysis of the effect of the combination of MOR202 and LEN on AMO-1cells in Example 4. Here NCI-H929 cells are tested and LEN alone has asignificant effect on the proliferation of NCI-H929 cells as shown inExample 5, therefore, the fractional product concept is utilized. Thefractional product concept was described in Ting-Chao Chou, TheoreticalBasis, Experimental Design, and Computerized Simulation of Synergism andAntagonism in Drug Combination Studies, Pharmacol Rev 58:621-681 (2006),which is incorporated by reference in its entirety. There Chou et al.states: If A and B each inhibits 60%, then it is oversimplification tosay that the additive effect is 84% inhibition. Based on the reasoningby Webb (1963), this type of problem can be solved by(1−0.6)(1−0.6)=0.16, 1−016=0.84. Chou and Talalay (1984) called it thefractional product method. This method will never lead to a combinationeffect exceeding 100% inhibition. Chou and Talalay (1984), however, havealso proved that this method has limited validity because it takes intoaccount the potency (e.g., fractional inhibition) but ignores the shapeof the dose-effect curve (e.g. hyperbolic or sigmoidal). The importanceof the “shape” in a dose-effect analysis is shown in FIG. 1. Chou andTalalay (1984) indicated that Webb's method is valid only when bothdrugs have hyperbolic curves (i.e., in simple Michaelis-Menten kineticswhen dose-effect curves are hyperbolic, i.e., m=1 in the median-effectplot) and is not valid when m does not equal 1, such as sigmoidal (m>1)or flat sigmoidal (m<1) curves. Furthermore, Webb's method is valid whenthe effects of two drugs are mutually nonexclusive (e.g., totallyindependent) and is not valid for mutually exclusive (e.g., similarmechanisms or modes of actions, as assumed for the classic isobologram,see below).

Clarke et al. was not utilized as Clarke is most suitable when onemonotherapy has a low effect.

See FIG. 12, the best fit curve was determined by removing the datapoints a) where the concentration of MOR202 was too low to have anyeffect and b) where the concentration was near saturation. At theappropriate date point, approx. 80% cell killing, the CI value is lessthan 1, supporting clear synergy.

Results

Applying the analysis of the Fractional Product Concept, LENsynergistically enhanced MOR202 activity in NCI-H929 cells in 6 out of 6experiments. Applying the analysis of Chou et al., LEN synergisticallyenhanced MOR202 activity in NCI-H929 cells in 6 out of 6 experiments.See Tables 7a-b, and 8a-b.

Example 7 MOR202 and LEN Alone and in Combination in NCI-H929 Bone LysisSCID Mouse MM Model

Materials

Lenalidomide (SYNthesis med chem; Shanghai, China; Lot no: ZHM-066-051).MOR202 (MorphoSys AG, Lot 100706-5KLE18). Vehicle control: Ora-Plus:Ora-Sweet SF (Paddock Laboratories, Minneapolis, Minn., USA, Lot no.9499528). SCID Mice (University of Adelaide, Waite Campus, Urrbaraie.SA, Australia, Strain C.B.-17-Igh-1^(b)-Prkdc^(scid)). NCI-H929 humanmultiple myeloma cells (see Table 1). RPMI 1640 cell culture medium,Foetal Bovine Serum (FBS), Mercaptoethanol, Hank's Balanced SaltSolution (HBSS) and penicillin-streptomycin from Invitrogen Australia(Mt Waverley, VIC, Australia); and Trypan Blue and glucose fromSigma-Aldrich (Castle Hill, NSW, Australia).

Methods

63 SCID mice were inoculated on Day (−7) orthotopically into the righttibia with 2.5×10⁶ NCI-H929 MM cells (in 5 μL) in order to induce bonelysis. Three days post inoculation (Day −4) 60 of the SCID mice wererandomized by body weight into the groups shown in Table 13, 10 mice pergroup. The dosing regimen is provided in Table 9. Lenalidomide (Groups Aand D) and Vehicle Control (Group C) treatments started on Day (−1).MOR202 treatments (Groups B and D) started on Day 0. Treatment continuedfor 6 weeks.

TABLE 9 Dosing regimen and Groups Group Compound Treatment Schedule ALenalidornide 50 mg/kg, p.o. in 10 mL/kg once daily for 6 weeks B MOR202 3 mg/kg, i.p., in 10 mL/kg 3 times weekly for 6 weeks C Vehicle Control10 mL/kg, p.o. once daily for 6 weeks (Ora-Plus:Ora- Sweet SF 1:1, w/w))D Lenalidomide/ 50 mg/kg, p.o. in 10 mL/kg once daily for 6 weeks MOR202 3 mg/kg, i.p., in 10 mL/kg 3 times weekly for 6 weeks CombinationMicroCT Scan was used to assess bone lysis and included 83-dimensionalanalysis comprising Total Bone Volume (TBV), Trabecular Bone Volume(Tb.BV), Trabecular Pattern Factor (Tb.Pf) and Structure Model Index(SMI). Table 10 defines each of these parameters. The results of each ofthe MicroCT Scan parameters are shown in Table 11. The Total Bone Volume(TBV) results are shown in FIG. 19.

TABLE 10 MicroCT Scan parameters Parameters: Definitions: Total BoneVolume Total cortical and trabecular bone volume within the (mm3) volumeof interest (cross-section). Trabecular Bone Trahecular bone volumewithin the volume of interest Volume (cross-section). {circumflex over( )}Trabecular Pattern Fragmentation index; An inverse index ofconnectivity Factor (Tb.Pf) with specific application to the trabecularhone. A lower Tb.Pf signifies better connected trabecular lattices whilehigher Tb.Pf means a more disconnected trabecular structure (I.e. morehone lysis). **Structure Model An indicator of the relative prevalenceof rods and Index (SMI) plates in a 3D structure such as the trabecularbone. This parameter is important in osteolysis of the bone Which ischaracterised by a transition from plate-like (normal) to rod-like(degradation) structures. An ideal plate, cylinder and sphere have SMIvalues of 0, 3 and 4 respectively. The higher the value, the more damagethere is.

TABLE 11 Results of the MicroCT Scan: Total Bone Volume (TBV),Trabecular Bone Volume (Tb · BV), Trabecular Pattern Factor (Tb · Pf)and Structure Model Index (SMI). Total Trabecular Trabecular Bone BonePattern Structure Volume Volume Factor Model (TBV) (Tb · BV) (Tb · Pf)Index Group Treatment Mouse ID mm⁻³ mm⁻² mm⁻¹ (SMI) ControlNon-inoculated 38045 2.748 0.244 15.354 1.756 reference 38596 2.8390.295 12.373 1.542 tibia* 39565 2.930 0.314 14.703 1.847 38325 2.9640.309 13.538 1.653 33746 2.751 0.293 13.270 1.624 38770 2.567 0.30713.025 1.645 37966 2.967 0.410 12.125 1.557 38604 3.087 0.327 11.9021.658 38023 2.775 0.270 18.005 1.889 38594 2.830 0.311 13.293 1.589 Mean2.846 0.308 13.759 1.676 SEM 0.047 0.014 0.583 0.937 A Lenalidomide,33150 1.604 0.107 24.329 2.679 50 mg/kg 38027 1.742 0.100 23.561 2.66738314 2.506 0.256 22.893 2.335 38446 2.466 0.213 28.280 2.560 385622.688 0.385 22.213 2.086 38626 2.869 0.293 30.739 2.619 38748 1.7860.114 24.016 2.562 39192 1.988 0.081 29.454 2.592 39364 1.741 0.15524.205 2.547 39512 2.007 0.219 38.360 3.125 Mean 2.140 0.192 26.8052.577 SEM 0.143 0.031 1.584 0.083 B MOR03087, 32094 2.233 0.190 27.0492.386 3 mg/kg 32548 2.893 0.310 15.631 1.818 33564 2.760 0.356 27.6312.423 38016 1.635 0.118 27.523 2.450 38023 2.681 0.248 17.887 1.86038510 1.838 0.260 21.405 2.461 38599 2.884 0.482 26.345 2.558 390863.068 0.566 20.327 2.247 39666 2.547 0.416 25.843 2.318 30715 2.1350.284 22.402 2.275 Mean 2.467 0.323 23.204 2.280 SEM 0.153 0.043 1.3650.079 C Vehicle 33090 1.821 0.159 26.537 2.714 Control 33131 1.863 0.13228.429 2.681 (Ora 33746 1.577 0.130 29.171 2.652 Plus:Ora 37966 1.8650.234 18.276 2.327 Sweet SF 38325 2.030 0.096 30.839 2.591 (1:1, w/w))38596 1.870 0.154 33.079 2.783 38604 1.904 0.232 23.234 2.607 387702.461 0.210 19.149 2.137 39426 1.556 0.184 21.846 2.348 39565 2.2350.256 24.545 2.365 Mean 1.918 0.179 25.510 2.521 SEM 0.087 0.017 1.5670.067 AB Lenalidomide, 32695 2.471 0.168 21.323 2.028 50 mg/kg/ 378542.527 0.265 15.938 1.758 MOR03087, 38276 3.212 0.220 20.046 2.197 3mg/kg 38550 2.833 0.186 17.907 1.892 38594 3.044 0.268 16.530 1.78738994 2.896 0.408 14.633 1.683 39256 2.308 0.304 24.513 2.280 395553.277 0.215 19.753 2.497 39677 3.205 0.548 12.313 1.799 39750 2.9610.281 14.981 1.752 Mean 2.868 0.286 17.794 1.967 SEM 0.105 0.036 1.1520.086

The analysis of each parameter for synergistic activity was performedaccording to theorem of Clarke et al. Table 12 shows the calculationsdone to determine synergy of the combination of MOR202 and lenalidomide.

TABLE 12 When POSITIVE EFFECT has a HIGH value: When POSITIVE EFFECT hasa LOW value: Antagonistic = (AB)/C < (A/C) × (B/C) Antagonistic =(AB)/C > (A/C) × (B/C) Additive = (AB)/C = (A/C) × (B/C) Additive =(AB)/C = (A/C) × (B/C) Synergistic = (AB)/C > (A/C) × (B/C) Synergistic= (AB)/C < (A/C) × (B/C) Trabecular Total BV Trabecular BV patternfactor Structural Model Index A 2.14 0.192 26.805 2.577 B 2.467 0.32323.204 2.28 C 1.918 0.179 25.51 2.521 AB 2.868 0.286 17.794 1.967 (AB)/C1.495307612 1.597765363 0.69753038 0.780245934 is bigger than is lessthan is less than is less than (A/C) × (B/C) 1.44 1.94 0.96 0.92  A =response to treatment LEN 50 mg/kg  B = response to treatment MOR202 3mg/kg  C = response to treatment vehicle AB = combination of treatments1 and 2

The numeric values shown in Table 12 are taken directly from theaverages shown in Table 11 for each of the parameters in each of theGroups. The Groups described as A, B, C and AB are the same treatmentgroups in both Tables 9, 11 and 12.

In Total Bone Volume (AB)/C is greater than (A/C)×(B/C) showing clearsynergism. In Trabecular pattern factor and Structural Model Index, asdescribed in Table 10, a lower value represents less bone lysis(efficacy in treatment), therefore, (AB)/C less than (A/C)×(B/C), showsclear synergism in both parameters.

Results

The inoculation of NCI-H929 multiple myeloma cells induced significantbone lysis in the tibiae of female SCID mice in this study, as indicatedby the measurement of bone lysis through microCT scanning. The degree ofbone lysis was significantly decreased in the tibia of mice treated withthe combination of MOR202 and lenalidomide as shown by microCT scanning.In each of the parameters of MicroCT Scan: Total Bone Volume (TBV),Trabecular Bone Volume (Tb.BV), Trabecular Pattern Factor (Tb.Pf) andStructure Model Index (SMI) the combination of MOR202 and lenalidomide(Group AB) showed clear synergy in the reduction of bone lysis caused bythe NCI-H929 multiple myeloma cells.

When the values in Table 11 are adjusted, so that the Control Group(Non-inoculated Contralateral Tibia without Tumour) is considered 0%bone lysis, and Group C (Vehicle Control (0.9% Sodium ChlorideInjection) is considerd 100% bone lysis, then MOR202 alone reduced bonelysis dose-dependently by up to 55% at 12 mg/kg compared to vehiclecontrol. LEN alone at 50 mg/kg inhibited bone lysis by 20%. Thecombination of 3 mg/kg MOR202 and 50 mg/kg LEN completely abolished bonelysis. These findings support a synergistic effect of combinationtherapy. In addition, there was a reduction (>90%) of M-protein serumlevels in the combination group, indicating a significant decrease oftumor load.

Example 8 MOr202 and Lenalidomide Alone and in Combination Against HumanNon-Hodgkin RAMOS Tumor in Female SCID Mice, Survival Model

Materials

Cyclophosphamide (Fluka, Buchs Switzerland, Lot. No. 07551661).Lenalidomide (SYNthesis Med Chem; Shanghai, China; Lot. #ZHM-066-051).MOR202 (MorphoSys AG, Lot 100706-5KLE18). Vehicle Control:Ora-Plus:Ora-Sweet SF, 1:1, v/v (SYNthesis Med Chem, Shanghai, China).SCID Mice (University of Adelaide, Waite Campus, Urrbaraie, SA,Australia, Strain C.B.-17-Igh-1^(b)-Prkdc^(scid)).

RAMOS cells (Oncodesign, Dijon Cedex, France) were cultivated inRPMI1640+20% heat inactivated alternate source FBS+1% Glutamax (Medium#2). Reagents for culture of RAMOS non-Hodgkin lymphoma cells wereobtained from the following suppliers: RPMI 1640 cell culture medium,FBS, Glutamax, HEPES, sodium pyruvate. HBSS, and penicillin-streptomycinfrom Invitrogen Australia (Mt Waverley, VIC, Australia); and Trypan Blueand glucose from Sigma-Aldrich (Castle Hill, NSW, Australia).

Methods

Sixty-eight female SCID mice were pre-treated with Cyclophosphamide (75mg/kg, i.p., twice daily) for two days prior to RAMOS cell inoculation(Day −5 and −4). On the day of inoculation (Day −3), all mice wereinoculated with 1×10⁶ RAMOS cells each intravenously into the tail vein.Sixty-four of the mice were randomised by body weight into eight groupsof eight. The dosing regimen for each group is shown in Table 13.

TABLE 13 Dosing regimen Group Compound Treatment Intended ScheduleActual Schedule A Lenalidomide 50 mg/kg, p.o., in Once daily (Day Day0-20 10 mL/kg 0-20) B MOR03087 1 mg/kg, i.v., in Twice weekly (Day Twiceweekly (Day 10 mL/kg 0, 4, 7, 11, 14 and 0, 4, 7, 11, 14 and 18) 18) CVehicle Control p.o., 10 mL/kg Once daily (Day Day 0-18(Ora-Plus:Ora-Sweet, 0-20) 1:1, v/v) AB Lenalidomide/ 100/1 mg/kg, Oncedaily/twice Day 0-13 and 16-20/ MOR03087 p.o./i.v., in 10 weekly (asabove) Day 0, 4, 7, 11, 14 mL/kg and 18The study continued for 98 days and the measured endpoint was survival.The results of each Group are shown in Table 14.

TABLE 14 Survival Number and time period for each group Number % ILS ofmice (based alive at on study median termina- Day of death(post-inoculation) death tion Median Range Mean 95% CI day) (day 98) A:LEN 100 22 18-23 21.4 19.8-23.0 10 0/7 mg/kg B: MOR202 51 35-65 49.641.9-57.3 155 0/8 1 mg/kg C: Vehicle 20 18-21 19.8 18.7-20.8 X 0/8control AB: Combo 65 32-98 66.5 41.9-91.2 225 3/8 LEN/MOR

Analysis for synergistic activity was performed according to theorem ofClarke et al., as described in Example 4. Table 15 shows thecalculations done in the determination of synergy of the combination ofMOR202 and lenalidomide.

TABLE 15 When POSITIVE EFFECT has a HIGH value: When POSITIVE EFFECT hasa LOW value: Antagonistic = (AB)/C < (A/C) × (B/C) Antagonistic =(AB)/C > (A/C) × (B/C) Additive = (AB)/C = (A/C) × (B/C) Additive =(AB)/C = (A/C) × (B/C) Synergistic = (AB)/C > (A/C) × (B/C) Synergistic= (AB)/C < (A/C) × (B/C) Median survival A 22 B 51 C 20 AB 65 (AB)/C3.25 is bigger than (A/C) × (B/C) 2.805  A = response to treatment withLEN 100 mg/kg  B = response to treatment with MOR202 1 mg/kg  C =response to treatment vehicle AB = combination of treatments A and B

The numeric values shown in Table 15 are taken directly from the mediansurvival days shown in Table 14 for each of the Groups. The Groupsdescribed as A, B, C and AB are the same treatment groups in Tables13-15.

The inoculation with RAMOS cells was lethal within a median time of 20days in the control group. The combination of MOR202 and lenalidomide,however, showed clear synergy in the increase in median survival days.

Example 9 Bortezomib Alone Inhibits Proliferation of Various MultipleMyeloma Cell Lines

The inhibitory effect of Bortezomib on proliferation of multiple myelomacells was analysed for multiple cell lines. Increasing amounts ofBortezomib (Velcade®, Lot: No.: #9AZSY00) were applied to AMO-1, LP-1,NCI-H929 and RPMI-8226 cells and incubated for 24 h, 48 h and 72 h.After incubation, period plates were analyzed for cell proliferation ina quantitative colorimietric XTT-based assay using the cellproliferation kit II (ROCHE, Cell Proliferation Kit II, Cat. No.:11465015001). For subsequent measurement, plates were subjected to TecanGenios Reader and absorbance at 492 nm was detected.

Cell proliferation of all tested cell lines was inhibited by Bortezomibwith an IC₅₀ concentration of 3.9 nM for AMO-1 cells, 6.1 nM for LP-1cells, 3.3 nM for NCI-H929 cells and 9.0 nM for RPMI-8226 cellsrespectively, as shown in FIG. 8.

Example 10 ADCC Using Combination of MOR202 and Bortezomib

Using the methods described in Example 4, the ADCC effect of combiningbortezomib and MOR202 was analyzed. Here, the target cells were treatedwith bortezomib prior to the treatment with MOR202. Both target cells.NCI-H929 and LP-1 cells were tested. The results are shown in FIGS. 9and 10. The enhancement in MOR202 activity by bortezomib was mediatedthrough a direct cytotoxic effect on MM cells.

Example 11 MOR202 and BOR Alone and in Combination in Human MultipleMyeloma NCI-H929 Bone Lysis SCID Mouse Model

Materials

Bortezomib (SYNthesis med chem., Shanghai, China, Lot no. #ZHM-066-054).Bortezomib was formulated in sterile 0.9% Sodium Chloride solution fordosing. MOR202 (MorphoSys AG, Lot 100706-5KLE18). Vehicle control: 0.9%Sodium Chloride Injection. SCID Mice (University of Adelaide, WaiteCampus, Urrbaraie, SA, Australia, Strain C.B.-17-Igh-1^(b)-Prkdc^(sid)).

Methods

63 SCID mice were inoculated on Day (−7) intra-tibially with 2.5×10⁶NCI-H929 MM cells in order to induce bone lysis. Three days postinoculation (Day −4) 60 of the SCID mice were randomized by body weightinto the groups shown in Table 16, 10 mice per group. The dosing regimenis provided in Table 16. Bortezomib (Groups A and AB) and VehicleControl (Group C) treatments started on Day (−1). MOR202 treatments(Groups B and AB) started on Day 0. Treatment continued for 6 weeks.

TABLE 16 Dosing regimen and Groups Group Compound Treatment Schedule ABortezomib 0.6 mg/kg, i.p., in 10 mL/kg twice per week B MOR202 3 mg/kg,i.p., in 10 mL/kg three times per week C Vehicle Control i.p., 10 mL/kgtwice per week (0.9% Sodium Chloride Injection) AB Bortezomib/MOR2020.6/3 mg/kg, i.p., in twice/three times per 10 mL/kg week, on alternatedays

MicroCT Scan was used to assess bone lysis and included a 3-dimensionalanalysis comprising Total Bone Volume (TBV), Trabecular Bone Volume(Tb.BV), Trabecular Pattern Factor (Tb.Pf) and Structure Model Index(SMI). Table 10 above defines each of these parameters. The results ofeach of the MicroCT Scan parameters are shown in Table 17. The resultsof the Total Bone Volume (TBV) is shown in FIG. 20.

TABLE 17 Results of the MicroCT Scan: Total Bone Volume (TBV),Trabecular Bone Volume (Tb · BV), Trabecular Pattern Factor (Tb · Pf)and Structure Model Index (SMI). Total Trabecular Trabecular Bone BonePattern Structure Volume Volume Factor Model (TBV) (Tb · BV) (Tb · Pf)Index Group Treatment Mouse ID mm⁻³ mm⁻² mm⁻¹ (SMI) Control: Referencetibia, 115898 2.771 0.400 12.097 1.525 Non-inoculated one mouse from116259 3.255 0.598 7.999 1.264 Contralateral Groups A, B, C 109482 3.1940.566 5.596 1.025 Tibia and AB) 107508 2.945 0.346 16.910 1.860 withoutAverage 3.041 0.477 10.650 1.419 Tumour SEM 0.112 0.062 2.481 0.179 ABortezomib, 101426 2.351 0.307 25.893 2.443 0.6 mg/kg, twice 1059492.025 0.191 26.044 2.374 per week, i.p. 107598 3.109 0.557 16.877 2.156109560 3.146 0.588 23.179 2.262 113302 1.790 0.067 32.463 2.700 1158361.893 0.076 33.152 2.981 116981 2.201 0.100 34.609 3.007 117585 1.6170.093 31.813 2.553 117750 2.300 0.284 27.147 2.337 117793 2.448 0.32923.582 2.273 Average 2.288 0.259 27.476 2.509 SEM 0.162 0.061 1.7560.094 Total Trabecular Trabecular Structure Bone Bone Pattern ModelVolume Volume Factor Index Group Treatment Mouse ID (TBV) (Tb · BV) (Tb· Pf) (SMI) B MOR202, 106446 1.924 0.082 27.363 2.546 3 mg/kg, three109482 1.987 0.388 19.479 2.079 times per week, 112220 2.155 0.39421.858 2.296 i.p. 113668 1.958 0.276 23.814 2.429 115187 2.080 0.44016.347 1.871 115956 2.207 0.460 19.748 2.193 116312 1.885 0.234 25.2122.368 116798 1.882 0.254 21.276 2.436 116944 1.937 0.276 24.031 2.368117773 1.862 0.160 25.056 2.511 Average 1.988 0.296 22.418 2.310 SEM0.038 0.039 1.044 0.066 C Vehicle Control 107097 1.619 0.248 22.7792.246 (0.9% Sodium 112122 1.608 0.178 26.514 2.505 Chloride 115971 1.6370.241 24.603 2.485 Injection), 116259 1.880 0.369 19.334 2.176 twice perweek, 116585 2.060 0.179 24.120 2.369 i.p. 116779 1.624 0.190 23.9092.417 117054 1.782 0.131 23.000 2.541 117110 1.838 0.281 22.602 2.312117242 1.919 0.281 21.162 2.193 117375 1.899 0.283 23.455 2.338 Average1.786 0.238 23.148 2.358 SEM 0.050 0.022 0.615 0.041 AB Bortezomib/MO107508 3.303 0.927 16.902 2.158 R202, 112625 4.254 1.661 3.000 0.7720.6/3 mg/kg, 113322 3.684 1.332 3.888 0.884 twice/three 116030 2.4220.192 30.272 2.542 times per week, 116198 3.537 1.037 8.023 1.217 i.p.116376 1.933 0.255 22.059 2.321 116520 2.793 0.654 22.439 2.336 1170773.402 0.658 7.207 1.241 117093 2.436 0.643 17.454 1.927 117135 3.0260.627 13.699 1.775 Average 3.079 0.799 14.494 1.717 SEM 0.220 0.1442.836 0.204

The Analysis of each parameter for synergistic activity was performedaccording to theorem of Clarke et al., as described in Example 4. Table18 shows the calculations done in the determination of synergy of thecombination of MOR202 and bortezomib.

TABLE 18 When POSITIVE EFFECT has a HIGHER value: When POSITIVE EFFECThas a LOWER value: Antagonistic = (AB)/C < (A/C) × (B/C) Antagonistic =(AB)/C > (A/C) × (B/C) Additive = (AB)/C = (A/C) × (B/C) Additive =(AB)/C = (A/C) × (B/C) Synergistic = (AB)/C > (A/C) × (B/C) Synergistic= (AB)/C < (A/C) × B/C) Group Total BV Trabecular BV Trabecular patternfactor Structural Model Index A 2.288 0.259 27.476 2.509 B 1.988 0.29622.418 2.31 C 1.786 0.238 23.148 2.358 AB 3.079 0.799 14.494 1.717(AB)/C 1.723964166 3.357142857 0.626144807 0.728159457 is bigger than isbigger than is less than is less than (A/C) × (B/C) 1.4259670521.53435492 1.149538246 1.042377527 A = response to treatment with BOR at0.6 mg/kg B = response to treatment with MOR202 at 3 mg/kg C = responseto treatment with vehicle 0.9% Sodium Chloride AB = combination oftreatments A and B

The numeric values shown in Table 18 are taken directly from theaverages shown in Table 17 for each of the parameters in each of theGroups. The Groups described as A, B, C and AB are the same treatmentgroups in Tables 16-18.

In Total Bone Volume and Trabecular Bone Volume, (AB)/C is greater than(A/C)×(B/C) showing clear synergism. In Trabecular pattern factor andStructural Model Index, as described in Table 10, a lower valuerepresents less bone lysis (efficacy in treatment), therefore, (AB)/Cless than (A/C)×(B/C), supports clear synergism in both parameters.

Results

The inoculation of NCI-H929 multiple myeloma cells induced significantbone lysis in the tibiae of female SCID mice in this study, as indicatedby the measurement of bone lysis through microCT scanning. The degree ofbone lysis was significantly decreased in the tibia of mice treated withthe combination of MOR202 and bortezomib as shown by microCT scanning.In each of the parameters of MicroCT Scan: Total Bone Volume (TBV),Trabecular Bone Volume (Tb.BV), Trabecular Pattern Factor (Tb.Pf) andStructure Model Index (SMI) the combination of MOR202 and bortezomib(Group AB) showed clear synergy in the reduction of bone lysis caused bythe NCI-H929 multiple myeloma cells.

When the values in Table 17 are adjusted, so that the Control Group(Non-inoculated Contralateral Tibia without Tumour) is considered 0%bone lysis, and Group C (Vehicle Control (0.9% Sodium ChlorideInjection) is considerd 100% bone lysis, then MOR202 alone reduced bonelysis dose-dependently by up to 55% at 12 mg/kg compared to vehiclecontrol, BOR alone at 0.6 mg/kg inhibited bone lysis by 40% and thecombination of a lower dose of 3 mg/kg MOR202 and 0.6 mg/kg BORcompletely abolished bone lysis. These findings support a synergisticeffect of combination therapy. In addition, there was a reduction (>90%)of M-protein serum levels in the combination group, indicating asignificant decrease of tumor load.

Example 12 MOR202 and Bortezomib Alone and in Combination Against HumanNon-Hodgkin RAMOS Tumor in Female SCID Mice, Survival Model

Materials

Cyclophosphamide (Fluka, Buchs Switzerland, WB10468). Bortezomib(SYNthesis med chem., Shanghai, China, Lot no. #ZHM-066-054). Bortezomibwas formulated in sterile 0.9% Sodium Chloride solution for dosing.MOR202 (MorphoSys AG, Lot 100706-5KLE18). Vehicle control: 0.9% SodiumChloride Injection. SCID Mice (University of Adelaide, Waite (Campus,Urrbaraie, SA, Australia, Strain C.B.-17-Igh-1^(b)-Prkdc^(scid)).

RAMOS cells (Oncodesign, Dijon Cedex, France) were cultivated inRPMI1640+20% heat inactivated alternate source FBS+1% Glutamax (Medium#2). Reagents for culture of RAMOS non-Hodgkin lymphoma cells wereobtained from the following suppliers: RPMI 1640 cell culture medium,FBS, Glutamax, HEPES, sodium pyruvate. HBSS, and penicillin-streptomycinfrom Invitrogen Australia (Mt Waverley, VIC, Australia); and Trypan Blueand glucose from Sigma-Aldrich (Castle Hill, NSW, Australia).

Methods

Fifty-five female SCID mice were pre-treated with Cyclophosphamide (75mg/kg, i.p., twice daily) for two days prior to RAMOS cell inoculation(Day −5 and −4). On the day of inoculation (Day −3), all fifty-five micewere inoculated with 1×10⁶ RAMOS cells each (in 100 μL) intravenouslyinto the tail vein. Forty-eight of the mice were randomised by bodyweight into six groups of eight. The dosing regimen for each group isshown in Table 19.

TABLE 19 Dosing regimen Group Compound Treatment Intended ScheduleActual Schedule A Bortezomib 0.6 mg/kg, i.p., in Day −1, 3, 6, 10, 13Day −1, 3, 6 and 13 10 mL/kg and 17 B MOR202 1 mg/kg, i.v., in Day 0, 4,7, 11, 14 Day 0, 4, 7, 11, 14 10 mL/kg and 18 and 18 C Vehicle Control(0.9% i.p., 10 mL/kg Day −1, 3, 6, 10. 13 Day −1, 3, 6, 13 and 17 Salinefor Injection) and 17 AB Bortezomib/MOR202 0.6/1 mg/kg, i.p./i.v., Day−1, 3, 6, 10, 13 Day −1, 3, 6, 13, 17 and in 10 mL/kg and 17/Day 0, 4,7, 20/Day 0, 4, 7, 11, 14 11, 14 and 18 and 18

The study continued for 98 days and the measured endpoint was survival.The results of each Group are shown in Table 20.

TABLE 20 Survival Number and time period for each group Number % ILS ofmice (based alive at on study median termina- Day of death(post-inoculation) death tion Median Range Mean 95% CI day) (day 98) A:BOR 0.6 19 18-20 19.1 18.4-19.8 −7 0/8 mg/kg B: MOR202 43.5 38-52 43.639.0-48.3 112 0/8 1 mg/kg C: Vehicle 20.5 20-22 20.8 20.0-21.5 x 0/8control AB: Combo 45 29-98 61.6 19.7-103.5 120 2/5 BOR/MOR

Analysis for synergistic activity was performed according to theorem ofClarke et al. Table 21 shows the calculations done in the determinationof synergy of the combination of MOR202 and bortezomib.

TABLE 21 When POSITIVE EFFECT has a HIGHER value: Antagonistic = (AB)/C< (A/C) × (B/C) Additive = (AB)/C = (A/C) × (B/C) Synergistic = (AB)/C >(A/C) × (B/C) Median survival A 19 B 43.5 C 20.5 AB 45 (AB)/C2.195121951 is bigger than (A/C) × (B/C) 1.966686496 A = response totreatment with BOR 0.6 mg/kg B = response to treatment with MOR202 1mg/kg C = response to treatment vehicle 0.9% Sodium Chloride AB =combination of treatments A and B

The numeric values shown in Table 21 are taken directly from the mediansurvival days shown in Table 20 for each of the Groups. The Groupsdescribed as A, B, C and AB are the same treatment groups in Tables19-21.

The inoculation with RAMOS cells was lethal within a median time of 20.5days in the control group. The combination of MOR202 and bortezomib,however, showed clear synergy in the increase in median survival days.Importantly, with the combination of MOR202 and bortezomib (Group AB), 2out of 5 mice survived for the duration of the study. This stronglysupports a synergistic finding of the combination of MOR202 andbortezomib.

It is to be understood that the description, specific examples and data,while indicating exemplary embodiments, are given by way of illustrationand are not intended to limit the present invention. Various changes andmodifications within the present invention will become apparent to theskilled artisan from the discussion, disclosure and data containedherein, and thus are considered part of the invention.

We claim:
 1. A method for treating multiple myeloma or non-hodgkins lymphoma in a subject in need thereof, wherein said method comprises administering to the subject a therapeutically effective amount of a combination of: (i) thalidomide, a thalidomide analog, lenalidomide, or pomalidomide and (ii) an antibody specific for CD38, comprising an HCDR1 region of sequence SYYMN (SEQ ID NO: 14) or GFTFSSYYMN (SEQ ID NO: 1), an HCDR2 region of sequence GISGDPSNTYYADSVKG (SEQ ID NO: 2), an HCDR3 region of sequence DLPLVYTGFAY (SEQ ID NO: 3), an LCDR1 region of sequence SGDNLRHYYVY (SEQ ID NO: 4), an LCDR2 region of sequence GDSKRPS (SEQ ID NO: 5), and an LCDR3 region of sequence QTYTGGASL (SEQ ID NO: 6).
 2. The method of claim 1, wherein said antibody comprises an HCDR1 region of sequence SYYMN (SEQ ID NO: 14).
 3. The method of claim 1, wherein said antibody comprises an HCDR1 region of sequence GFTFSSYYMN (SEQ ID NO: 1).
 4. The method of claim 1, wherein said antibody comprises a variable heavy chain of the sequence: (SEQ ID NO: 10) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVS GISGDPSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DLPLVYTGFAYWGQGTLVTVSS;

and a variable light chain of the sequence: (SEQ ID NO: 11) DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYG DSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQTYTGGASLVF GGGTKLTVLGQ.


5. The method of claim 1, wherein said antibody is an IgG antibody.
 6. The method of claim 1, wherein said antibody comprises an IgG1 Fc region.
 7. The method of claim 1, wherein said antibody comprises a modified Fc region, wherein said modification enhances ADCC activity.
 8. The method of claim 1, wherein said combination comprises said antibody and thalidomide.
 9. The method of claim 8, wherein said cancer is multiple myeloma.
 10. The method of claim 1, wherein said combination comprises said antibody and lenalidomide.
 11. The method of claim 10, wherein said cancer is multiple myeloma.
 12. The method of claim 1, wherein said combination comprises said antibody and pomalidomide.
 13. The method of claim 12, wherein said cancer is multiple myeloma.
 14. The method of claim 1, wherein said antibody and said thalidomide, thalidomide analog, lenalidomide, or pomalidomide are administered separately.
 15. The method of claim 8, wherein said antibody and said thalidomide are administered separately.
 16. The method of claim 10, wherein said antibody and said lenalidomide are administered separately.
 17. The method of claim 12, wherein said antibody and said pomalidomide are administered separately.
 18. The method of claim 9, wherein said antibody and said thalidomide are administered separately.
 19. The method of claim 11, wherein said antibody and said lenalidomide are administered separately.
 20. The method of claim 13, wherein said antibody and said pomalidomide are administered separately. 